Hydrological regimes are being perturbed under climate change due to the regional expression of the water cycle across the globe, leading to alterations in the spatial and temporal distribution of water near the Earth's surface. Water is a critical resource for plant ecosystems, and hydrological limitations on vegetative health are particularly complex. To anticipate how subsurface water availability may evolve in the future and affect the dynamics of plant water source usage, as well as the health and functioning of vegetation in various biomes, we need a robust, quantitative framework for linking water availability to past plant water use, which is constrained by historical data. Here, we outline the Identification of Source-water Oxygen isotopes in trees Toolkit (ISO-Tool), designed to retrospectively investigate the dynamics of tree water uptake. ISO-Tool utilizes tree-ring isotopes (δ 18 O) combined with a biomechanistic fractionation model to retrodict the δ 18 O of water utilized during any period of growth. Through comparisons with measured δ 18 O in local water sources, climatic, and hydrological variables, ISO-Tool can reconstruct and inform on past ecohydrological interactions. We provide an overview of the modeling components and data requirements necessary to constrain the retrodictions of source-water δ 18 O. We demonstrate the utility and efficacy of ISO-Tool for three riparian field sites characterized by differences in climatic, geomorphic, and hydrologic complexity. We also state that ISO-Tool can be applied to a range of vegetated environments where distinct isotopic endmembers exist. We present a set of tool groups, which can be applied adaptively, ensuring that scientific progress in understanding retrospective ecohydrology can be made, even under varying degrees of data availability.